Studies of Collagen Gene Regulation in Two Murine Models

描述(由申请者提供)：此方案将使用两个鼠标 突变是硬皮病、紧绷皮肤(Tsk)和紧绷皮肤的模型2 (Tsk2)。这两种突变都表现出胶原蛋白过度积累和其他 皮肤中的细胞外基质成分，这是人类的一个特征 疾病。拟议研究的长期目标是利用 这两个突变与几个转基因小鼠品系相结合 剖析疾病发病的分子机制的实验工具。 提出了具体的实验来鉴定涉及到的基因。 真皮成纤维细胞细胞外基质合成的调控。二 试验性战略是有计划的，并包含三个具体目标。 具体目标1侧重于确定I型顺式作用元件 突变体中增加胶原基因表达所需的胶原基因 真皮成纤维细胞。定义“纤维化”的特定成分将提供一个基础 用于鉴定与这些DNA相互作用的交易因子 片段以增加胶原基因的表达。这些元素将被定义 通过研究携带不同基因的原代猫报告基因的表达 5‘启动子区域的片段以及第一个 内含子。每种转基因的表达将在皮肤样本中进行评估。 分离自TSK、Tsk2和正常小鼠。此外，转基因表达将是 从这些分离的皮肤外植体培养的真皮成纤维细胞中进行测量 老鼠。为了产生实验小鼠，将TSK和Tsk2突变小鼠杂交 转基因小鼠携带不同的胶原转基因构建物。一个 胶原第一内含子在转录上调中的潜在作用 在TSK和Tsk2突变(我们的 初步数据)以及硬皮病真皮成纤维细胞。以特定的目标 2胶原蛋白第一内含子在调控胶原蛋白转录中的作用 基因与Tsk和Tsk2纤维化皮肤表型的发展 下定决心。对于这些实验，首先在颈椎中进行有针对性的缺失 将使用内含子。这个实验模型有一个独特的特点 允许测定由CO11a1基因产生的 同一RNA制剂中缺失和正常等位基因。进一步说，这一基因 系统允许在以下环境中监视基因表达 内源基因。第二个实验方向涉及识别 与胶原水平升高相关的真皮成纤维细胞 采用微阵列分析的生产。中概述的实验计划 具体目标3包括开发试剂以分离特定的 突变动物和正常动物真皮成纤维细胞的群体培养 根据它们的胶原基因表达。这将通过雇佣员工来实现 一种胶原启动子绿色荧光蛋白报告转基因已被记录展示 TSK和Tsk2真皮成纤维细胞的高表达 突变的小鼠。真皮成纤维细胞表达该蛋白的流式细胞术分析 转基因将允许根据它们的水平来分离细胞群体 胶原蛋白的表达。RNA将从高胶原蛋白和低胶原蛋白中提取 产生胶原蛋白的细胞群。这些RNA将被用于 利用基因芯片技术筛选高胶原蛋白差异表达基因 与低胶原生成细胞相比，VISA也是如此。它是 预计在这一实验范式中确定的基因将允许 解剖与血管紧张素转换酶相关的分子通路 硬皮病，并可能导致控制细胞外基质的治疗 新陈代谢。

DESCRIPTION (provided by applicant): This proposal will utilize two mouse mutations that are models for scleroderma, tight skin (Tsk) and tight skin 2 (Tsk2). Both mutations display excessive accumulation of collagen and other extracellular matrix components in the skin, a hallmark feature of the human disease. The long range of objective of the proposed research is to utilize these two mutations combined with several lines of transgenic mice as experimental tools to dissect molecular mechanisms of disease pathogenesis. Specific experiments are proposed for the identification of genes involved in the regulation of extracellular matrix synthesis in dermal fibroblasts. Two experimental strategies are planned and are encompassed in three specific aims. Specific aim 1 focuses on identifying cis-acting elements in the type I collagen gene required for the increased production of Collal mRNA in mutant dermal fibroblasts. Defining "fibrotic" specific elements will provide a basis for the identification of the transacting factors that interact with these DNA segments to increase Collagen gene expression. These elements will be defined by studying the expression of Collal CAT reporter transgenes bearing various segments of the 5' promoter region as well as specific deletions of the first intron. The expression of each transgene will be evaluated in skin samples isolated from Tsk, Tsk2 and normal mice. Also, transgene expression will be measured in dermal fibroblasts cultured from skin explants isolated from these mice. To generate experimental mice, Tsk and Tsk2 mutant mice will be crossed with transgenic mice bearing the various collagen transgene constructs. A potential role of the Collal first intron in the upregulation of transcription of the Collal gene has been shown with the Tsk and Tsk2 mutations (our preliminary data) as well as in scleroderma dermal fibroblasts. In specific aim 2 the role of the Collal first intron in regulating transcription of the Collal gene and the development of the Tsk and Tsk2 fibrotic skin phenotype will be determined. For these experiments a targeted deletion in the Collal first intron will be employed. This experimental model has a unique feature permitting the determination of the levels of Co11a1 mRNA produced by the deleted and normal allele in the same RNA preparation. Further this genetic system allows the monitoring of gene expression in the context of the endogenous gene. A second experimental direction involves identifying genes in dermal fibroblasts that are associated with elevated levels of collagen production employing micorarray analysis. The experimental plan outlined in specific aim 3 includes the development of reagents to isolate specific populations of dermal fibroblasts cultured from both mutant and normal animals based on their collagen gene expression. This will be accomplished by employing a collagen promoter GFP reporter transgene that has been documented to display elevated expression in dermal fibroblasts isolated from both Tsk and Tsk2 mutant mice. Flow cytometric analysis of dermal fibroblasts expressing this transgene will permit the isolation of cell populations based on their level of collagen expression. RNA's will be extracted from high collagen and low collagen producing cell populations. These RNA's will be utilized in a microarray analysis to identify genes differentially expressed in high collagen producing cells compared to low collagen producing cells and visa versa. It is anticipated that genes identified in this experimental paradigm will permit the dissection of molecular pathways that are involved with the onset of scleroderma and potentially lead to therapies to control extracellular matrix metabolism.